The red imported fire ant, Solenopsis invicta Buren, is a highly invasive eusocial insect pest that threatens native biodiversity, agriculture, and human health. The innate immune system and intricate social immune responses of S. invicta pose challenges to the development of effective control strategies. MicroRNAs (miRNAs) play critical roles in the post-transcriptional regulation of gene expression, which influences various biological processes, including immunity and host-pathogen interactions. While the miRNA-mediated response of insects to pathogens has been extensively studied in solitary insects, little is known about the innate immune responses of individual members within a colony. To address this gap, we constructed small RNA libraries from Metarhizium anisopliae-infected S. invicta workers and investigated the temporal dynamics of miRNA-mediated immune responses to the entomopathogen. Several differentially expressed miRNAs were identified, and they were found to regulate genes involved in the Toll, IMD, and melanization immune pathways. Quantitative real-time PCR (qRT-PCR) was employed to analyze the spatiotemporal dynamics of key miRNAs/target genes, specifically miR-71/ModSP1-Relish and miR-7/Lysozyme2-Serine protease7. A dual luciferase assay (in vitro) was performed to validate the interactions between miRNAs and their target genes. Overexpression of miR-71 and miR-7 (via miRNA mimics) efficiently suppressed their target genes, impaired the antifungal immune response of S. invicta and increased the susceptibility to M. anisopliae infection compared to controls. Furthermore, RNA interference-based gene silencing elucidated the roles of these immune genes in regulating fungal susceptibility, thus providing vital clues for developing virulent and effective mycoinsecticides using modern genetic engineering tools.